Figure 1.

Tissue distribution and subcellular localization of R-Ras. (A) Immunohistochemistry analysis of mouse intestine with preimmune (left) or anti-R-Ras serum (right). Bound antibodies were detected with diaminobenzidine tetrahydrochloride, and the nuclei were counterstained with hematoxylin. Note the staining of the smooth muscle cell layer. (B) Immunohistochemistry of the mouse adrenal gland and pancreas. Note the staining of the adrenal medulla and Langerhans' islands. MDCK cells were fixed in 3.7% formaldehyde and methanol (C) or 4% paraformaldehyde (D) as described in the text, stained with the anti-R-Ras serum and the Alexa 488-coupled anti-rabbit IgG antibody, and observed with a confocal fluorescent microscope. Twenty-five XY images were obtained from the bottom to the top of the cells to prepare the stacked XY image. Cross sections are also shown at the dotted lines. Bar, 10 μm. (E) Immunoelectron micrograph of a MDCK cell stained with anti-R-Ras serum before detection with anti-rabbit IgG labeled with 10-nm gold particles. Bar, 100 nm. (F) Immunoelectron micrograph of a MDCK cell expressing an endosomal marker protein, AcGFP-Endo. Cells were double-stained with anti-R-Ras rabbit serum and anti-GFP mouse mAb JL-8, which were detected with anti-rabbit IgG labeled with 5-nm gold particles and anti-mouse IgG labeled with 10-nm gold particles, respectively. Inset depicts the magnified image of the gold particles. Bar, 100 nm.